General Histology & Molecular Techniques Flashcards
Explain Principles of Immunohistochemistry
Involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
The indirect method involves an unlabeled primary antibody (first layer) that binds to the target antigen in the tissue and a labeled secondary antibody (second layer) that reacts with the primary antibody. As mentioned above, the secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to the fluorescent or enzyme reporter.
Explain how to perform a Gram stain
A Gram stain is a method of staining tissues, in order classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria.
Based on differences in cell walls of bacteria (thickness of the peptidoglycan layer)
Method:
1) a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they don’t rinse out during the staining procedure.
2) Addition of iodine, which binds to crystal violet and traps it in the cell
3) Rapid decolorization with ethanol or acetone
4) Counterstaining with safranin or carbol fuchsin
Ziehl–Neelsen staining is a type of acid-fast stain and is used to identify acid-fast organisms, mainly Mycobacteria.
Summary of acid-fast stain (Ziehl–Neelsen stain)
1) Primary dye - Carbol fuchsin
2) Decolorizer - Acid alcohol
3) Counter stain - Methylene blue/malachite green
Explain how you would validate a new IHC stain in your laboratory
- Work with your senior scientist who does the IHC
- Follow the manufacturers protocols / data sheet
- Antigen retieval relies on temperature, pH and set processing time
- Use of both positive and negative control tissue (either commerical or in house)
- Several practice runs to optimise antibody before putting into general use
Explain how DIFL works
Direct immunofluorescence is a technique for detecting deposits of immunoglobulins and complement proteins in biopsies of skin, kidney and other organs.
Primary, or direct, immunofluorescence uses a single antibody that is chemically linked to a fluorophore. The antibody recognizes the target molecule and binds to it, and the fluorophore it carries can be detected via microscopy.
Secondary, or indirect, immunofluorescence uses two antibodies; the unlabeled first (primary) antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore, recognises the primary antibody and binds to it. Multiple secondary antibodies can bind a single primary antibody. This provides signal amplification by increasing the number of fluorophore molecules per antigen.
In skin: IgG, IgM, IgA, C3 and fibrin (frozen, perilesional)
How do you decalcify tissue?
Bone decalcification is the softening of bones due to the removal of calcium ions
There are two categories of decalcifying agents for removing calcium ions:
1) Chelating agents e.g Ethylenediaminetetraacetic acid (EDTA).
- chelating agents take up / absorb the calcium ions.
- slow but better preservation of cytologic detail, better for IPX and molecular tests
2) Acids -weak acids e.g 10% formic acid, strong acids e.g HCL
- acids help produce a solution of calcium ions
- fast but risk of losing cellular detail (esp. if over decalcify), can lose antigenicity for IPX and destroy DNA for molecular tests.
** ratio 20 : 1; change every 24 hours.
Explain principles of a DQ stain.
Diff-Quik is a commercial Romanowsky stain variant used to rapidly stain a variety of pathology specimens. Based on a modification of the Wright-Giemsa stain (faster).
Steps:
1: Fixative: 100% ethanol / methanol
2: Diff-Quik solution I (eosinophilic) Xanthene dye (Eosin Y), pH buffer
3: Diff-Quik solution II (basophilic) Thiazine dye, methylene blue, azure A, pH buffer
What special stains are commonly used in skin biopsies?
PAS - fungi, basement membrane
Other bug stains: Fite, Warthin-Starry, GMS, Gram
Hales - dermal mucin
Perls - iron deposition
Masson - collagen deposition, fibrosis, perforating disorders
EVG - elastin fibres
CR - amyloid deposition
What are the components of formalin in your lab
10% neutral buffered formalin is a 1:10 dilution of 100% formalin in water i.e. 1 part 100% formalin to 9 parts water with sodium phosphate added as a buffer.
100% formalin contains ~40% formaldehyde therefore a 1:10 dilution contains about 4% formaldehyde.
If in doubt check manufacture data sheet (by safety cabinet) or ask lead scientist.
What is in IMDM or RPMI?
IMDM and RPMI are synthetic cell culture media.
Like all cell culture media they contain nutrients to ensure cell survival ex vivo eg. amino acid, peptides, minerals, vitamins and buffering agents.
Exact details check manufacturers data sheet.
Explain Principles of FISH
Fluorescent in situ hybridisation (FISH)
Molecular cytogenetic technique that uses a fluorescent probe that binds specifically to part of a nucleic acid sequence.
Probe = single strand of DNA or RNA that is complementary to the region of interest.
Fluorecent microscope = used to identify where fluorescent probes are bound to chromosomes
Can be used to DNA, RNA, tissue (fresh frozen, FFPE) and circulating tumour DNA
5 STEPS
- DNA unmasking
- Probe and target denaturation
- Probe and target hybridisation
- Detection of target
- Image analysis under fluorescent microscope
Advantages of FISH
Relatively quick
Large number of cells can be scored over a short period
High sensitivity and specificity
Cytogenetic data can be derived from non dividing or terminally differentiated cells
Disadvantages of FISH
Relatively expensive
Need to know target of interest
Sample may not be suitable: degrade, necrotic
Cannot detect small mutations / rearrangements (low end of resolution ~ 15mb)
Fluorescence fades over few weeks - need to photograph!
Principles of Flow Cytometry
Flow cytometry is a technique for the rapid analysis of a statistically significant population of cells at a single cell level.
Usually a single cell suspension, labelled with fluorescent markers or antibodies and passed through a laser beam one cell at a time.
The light absorbed and emitted as wavelengths is can be sensed by photomultiplying tubes (PMT’s) and converted into data that is produced via computer.
What are Barr Bodies?
Barr bodies are an inactive X chromosome present in a cell which has more than one X chromosome.
Lyonisation = process in which a cell with multiple X chromosomes, all but one are inactivated during embryogenesis.
Therefore females with two X chromosomes will have 1 Barr body per somatic cell.
Males with one X chromosome will have 0 Barr bodies per somatic cell.
Can use Barr bodies to determine if tissue comes from a male or a female.
Common Uses for Flow Cytometry
Immunophenotyping lymphomas and leukaemias
Histocompatibility cross matching
Transplant rejection
Auto and alloimmune disorders
Reticulocyte enumeration
Feto maternal haemorrhage quantification
Advantages of Flow Cytometry
Can measure large number of parameters on the same sample
Fast - can collect information about a large number of cells very quickly
Cytospin taken at time of preparing sample can be used for morphologic assessment / checking concordance
Disadvantages of Flow Cytometry
Expensive
Sophisticated instrument, requires experienced and trained scientists to run and interpret results
Sample may not be representative
Explain Principles Of ISH
In Situ Hybridisation
A method for detecting nucleic acid sequences (RNA / DNA) in histologic section by applying a complementary strand of nucleic acid to which a reporter molecule is attached.
Types of Probes:
dsDNA, ssDNA, RNA, synthetic oligonucleotides
Labelling Techniques:
radioactive isotope, biotin, fluorescent dye
What is the Urovysion Test For?
FISH test using enumeration probes for the centromere regions of chromosomes 3, 7, 17 and probe for p16 (9q21).
Urothelial carcinoma is characterised by aneuplody with typically gains of chromosomes, 3, 7 and 17 and loss of p16.
“Positive” FISH result can also be seen in:
Primary bladder adenocarcinoma
SCC of the bladder
Prostate adenocarcinoma
Colorectal carcinoma
- > correlate wtih clinical, radiologic findings as well as urine cytology +/- histology
- > particularly given prostate cancer and colorectal cancer can secondarily involve the bladder
Explain PD1 / PDL1 Testing
The PD1 receptor and its ligands (PDL1 / PDL2) are part of a group of checkpoint inhibitors that act as coinhibitory factors in the cancer immunity cycle.
Tumour cells can hijack this process in order to evade detection by the immune system.
- > PDL1 is overexpressed by tumour cells
- > binds to PD1 on activated T-cells
- > leads to inhibition of cytotoxic T cells
Monoclonal antibodies against PDL1 and PD1 (eg pembrolizumab) can allow the immune system to recognise the tumour cells as non-self meaning cytotocic T cells and NK cells can attack tumour cells.
Detection of PDL1 expression by IPX determines if disease will be treatment responsive.
In NZ PDLI testing can be offered for NSCLC, TNBC, Urothelial ca, Gastric ca and H&N SCC.
Explain Principles of PCR
PCR = polymerase chain reaction
lab technique for rapid production or amplification of millions to billions of copies of a specific segment of DNA which can then be studied in more detail
Involves using short synthetic DNA fragments called primers to select the segment of genome to be amplified
Then multiple rounds of DNA synthesis to amplify that sequence
Need: DNA, primers, TAQ polymerase, nucleotides and buffer mix in a PCR tube and a thermal cycler
Steps: Denaturing, annealing and extension
Steps for Preparing EM
- Small 1mm cube of tissue
- Primary fixation in glutaraldehyde
- Secondary fixation in osmium tetroxide
- Rinse and dehydration steps (ethanol)
- Dry with liquid CO2
- Embed by infusing with resin and cut thin (70nM) sections with diamond blade or cleaved glass
- Post stain and image
What are these structures?
Birbeck granules - Langerhan cells
What does this picture show?
Primary ciliary dyskinesia
Circle and arrow = loss / reduction of dynein arms
What does this cardiac biopsy show?
Fabry disease! (dense lamellar bodies or Zebra bodies)
What does this renal biopsy show?
Amyloid!
Name this testicular tumour
Leydig cell tumour with Reinke crystals
Name this kidney tumour
Juxtaglomerular cell tumour
Rhomboid shaped pro-renin granules in cytoplasm
Pleural tumour in adult male
Malignant mesothelioma with microvilli (and tight junctions)
Adrenal tumour in a 2 year old
Neuroblastoma with dysmorphic synaptic vesicles containing neurosecretory granules
BRAF gene
BRAF is a proto-oncogene within the MAPK pathway
Following a mutation within the gene it becomes an oncogene and becomes constitutively active driving cell signalling pathways and cell proliferation
BRAF mutations seen in wide range of cancers: PTC, LCH, ECG, HCL, melanoma, lung, CRC
Presence of BRAF mutation means that BRAF inhibitor therapy may be an option for the patient
Also if a BRAF mutation is present then it is unlikely that TKI’s for upstream targets e.g EGFR will be effective
EGFR
EGFR is a receptor tyrosine kinase involved in cell signalling pathways that regulate cell division and survival
Amplification and mutations in EGFR gene lead to a constitutively active tyrosine kinase receptor with sustained cell signalling and proliferation
EGFR is implicated in a number of tumours (e.g lung adenocarcinoma and high grade gliomas) and is targetable
Long term use of TKI’s lead to drug resistance e.g. EGFRT790M - identifying this means patient can switch to 3rd generation TKI
APC gene
APC is a tumour suppressor gene that codes for the APC protein which is a negative regulator of the cell cycle
APC controls ß-catenin concentrations and interacts with e-cadherin as part of Wnt pathway
Germline loss = FAP syndrome, attenuated FAP, Gardner, Turcot
Somatic mutation = colorectal cancer
Interpret Gene Sequencing Data / Electropherogram
Typical electropherogram covers a single PCR product (average # of nucleotides 200 - 600) depending on size of gene being sequenced
Comparative analysis of patient sample and a DNA sample without a mutation
Each peak represents a single nucleotide in the DNA sequence.
Each nucleotide has a differnt colour (A = green, T=red. C=blue, G= black)
Sequencing analysis usually performed by software
Comparative Genomic Hybridisation
Molcular cytogenetic technique for analysing copy number variations relative to ploidy level in the DNA of a test sample compared with a reference sample
Aim to compare 2 genomic DNA samples from 2 sources because they are suspected to have differences in gains or losses of chromosomes / subchromosomes
Array CGH with competitive FISH has a resolution of 5 - 10 kilobases
Next Generation Sequencing
Sequencing of multiple specimens in parallel
Targeted gene panel, whole exome, or whole genome
Multiple commercially available platforms with varying engineering configurations and forms of sequencing chemistry
Basic Steps:
- Build library by extracting DNA and amplification by PCR
- Sequencing
- Data extraction and bioinformatics
